Targeting WSB1 and pVHL to treat cancer

ABSTRACT

Materials and methods for treating cancer (e.g., by reducing metastasis) are provided herein. For example, materials and methods for treating cancer by targeting WSB1 and/or pVHL are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 62/157,554, filed on May 6, 2015.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA189666, awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

This document relates to materials and methods for treating cancerand/or inhibiting cancer metastasis, such as materials and methods fortreating cancer by targeting WSB1 and/or pVHL.

BACKGROUND

The loss of pVHL leads to von Hippel-Lindau disease, which ischaracterized by development of tumors that can include renal clear-cellcarcinomas (RCCs) and other highly vascularized tumors (Kaelin, Ann RevPathol 2:145-173, 2007; Kaelin, Nature Rev Cancer 2:673-682, 2002; andLatif et al., Science 260:1317-1320, 1993). pVHL is thesubstrate-recognition component of a cullin RING ubiquitin ligasecomplex also that includes elongin B, elongin C, Rbx1 and Cul2 (Deshaiesand Joazeiro, Ann Rev Biochem 78:399-434, 2009; and Kaelin, Nature RevCancer 8:865-873, 2008). pVHL's main function as an E3 ligase is totarget HIF-1α for degradation during normoxia (Kaelin 2002, supra). Theloss of pVHL results in constitutive activation of HIF-1α, which acts asan important transcription factor for target genes such as VEGF, GULT1,CAIX, and HK2 (Gossage et al., Nature Rev Cancer 15:55-64, 2015). As aconsequence, HIF-1α induces metabolic adaptation and promotes tumorgrowth, invasion, migration, metastasis, and angiogenesis through upregulation of its target genes (Gordan and Simon, Curr Opin Genet Devel17:71-77, 2007; Semenza, Trends Mol Med 7:345-350, 2001; and Semenza,Oncogene 29:625-634, 2010).

pVHL is ubiquitously expressed in normal tissues and cell types (Los etal., Laboratory Investigation; A Journal of Technical Methods andPathology 75:231-238, 1996). Loss of the VHL gene and germline mutationsare important mechanisms of pVHL down regulation in various cancers(Gossage et al., supra), but the regulation of pVHL at theposttranscriptional level remains underexplored. pVHL may be regulatedthrough the ubiquitin—proteasome pathway (Chen et al., Biology of theCell/Under the Auspices of the European Cell Biology Organization105:208-218, 2013; Jung et al., Nature Med 12:809-816, 2006; andPozzebon et al., Proc Natl Acad Sci USA 110:18168-18173 2013), althoughthe identity of pVHL's E3 ligase is not clear.

WD repeat and SOCS box-containing protein 1 (WSB1) has been classifiedas a substrate recognition subunit of the ECS (ElonginB/CCul2/5-SOCS)ubiquitin ligase complexes (Vasiliauskas et al., Mech Devel 82:79-94,1999). WSB1 harbors seven WD40 repeats and a SOCS box (Choi et al., JBiol Chem 283:4682-4689, 2008). The expression of WSB1 positivelycorrelates with tumor incidence in cancers such as pancreatic cancer,hepatocellular carcinoma, and salivary gland tumor (Archange et al.,PloS One 3:e2475, 2008; Rhodes and Chinnaiyan, Nature Genet37(Suppl):531-37, 2005; Silva et al., Bioinformatics 27:3300-3305, 2011;and Tong et al., FEBS Lett 587:2530-2535, 2013). WSB1 also is a targetof HIF-1 (Tong et al., supra).

The cellular function of WSB1 has not been well studied. WSB1 canmediate homeodomain-interacting protein kinase 2 (HIPK2) ubiquitination,resulting its proteasome degradation (Choi et al., supra). Following DNAdamage, WSB1-mediated ubiquitination of HIPK2 is blocked, resulting inHIPK2 stabilization. HIPK2 in turn phosphorylates p53 at Ser46, which isimportant for activating proapoptotic gene expression (Puca et al.,Oncogene 29:4378-4387, 2010). WSB1 overexpression has been shown topromote pancreatic cancer cell proliferation (Archange et al., supra).However, this effect is unlikely due to inactivation of HIPK-p53pathway, as the pancreatic cancer cell line used in the study containsmutant p53. Thus, WSB1 may promote cancer cell proliferation throughother p53-independent mechanisms.

SUMMARY

This document is based, at least in part, on the discovery that WSB1 isa negative regulator of pVHL through WSB1's E3 ligase activity.Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomaldegradation, thereby stabilizing HIF under both of normoxia and hypoxiaconditions. As a consequence, WSB1 upregulates the expression ofHIF-1α's target genes and promotes cancer invasion and metastasisthrough its effect on pVHL. Consistent with this, WSB1 protein levelsare negatively correlated with pVHL levels and metastasis-free survivalin clinical samples. The work described herein reveals a new mechanismof pVHL's regulation, by which cancer acquires invasiveness andmetastatic tendency.

In one aspect, this document features a method for treating a cancerpatient by administering to the cancer patient an agent that reduces theactivity of WSB1. The agent can be an inhibitory nucleic acid (e.g., ashRNA) targeted to a WSB1 nucleic acid that is endogenous to the cancerpatient, or can be an antagonistic antibody to WSB1. The method caninclude administering to the cancer patient a composition containing theagent and a pharmaceutically acceptable carrier.

In another aspect, this document features a method for inhibitingmetastasis of a tumor in a cancer patient by administering to the cancerpatient an agent that reduces the activity of WSB1. The agent can be aninhibitory nucleic acid (e.g., a shRNA) targeted to a WSB1 nucleic acidthat is endogenous to the cancer patient, or can be an antagonisticantibody to WSB1. The method can include administering to the cancerpatient a composition containing the agent and a pharmaceuticallyacceptable carrier.

In another aspect, this document features a method for treating a cancerpatient by administering to the cancer patient an agent that increasesthe activity of pVHL. The agent can be a nucleic acid encoding a pVHLpolypeptide (e.g., a nucleic acid operably linked to a promoter thatdrives expression of the nucleic acid), a pVHL polypeptide, or anagonistic antibody to pVHL. The method can include administering to thecancer patient a composition containing the agent and a pharmaceuticallyacceptable carrier.

This document also features a method for inhibiting metastasis of atumor in a cancer patient by administering to the cancer patient anagent that increases the activity of pVHL. The agent can be a nucleicacid encoding a pVHL polypeptide (e.g., a nucleic acid operably linkedto a promoter that drives expression of the nucleic acid), a pVHLpolypeptide, or an agonistic antibody to pVHL. The method can includeadministering to the cancer patient a composition containing the agentand a pharmaceutically acceptable carrier.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1E demonstrate that WSB1 is positively related with metastasisand expression of HIF target genes. FIG. 1A is a table containing ananalysis of differentially expressed genes by WSB1 expression levels inlung adenocarcinoma patients using INGENUITY pathway analysis. FIG. 1Bis a series of graphs plotting levels of WSB1 expression in primary andmetastatic melanoma (left panel), prostate cancer (center panel), andurinary bladder cancer (right panel) (GEO data sets GSE840, GSE6916, andGSE3167), demonstrating that WSB1 is highly expressed in humanmetastatic cancers. FIG. 1C is a series of Kaplan-Meier graph for humanbreast (left and center panels) and colon (right panel) cancer patients,stratified according to high or low expression levels of WSB1 (usingPROGgene). FIG. 1D is a table listing potential HIF-1α target genes thatmay be associated with WSB1 expression levels in lung adenocarcinomapatients. FIG. 1E is a series of graphs plotting the co-relation betweenWSB1 and HIF target gene expression in metastatic melanoma (top row),prostate (middle row), and breast (bottom row) cancer patients (GEO dataset).

FIG. 2 is a graph plotting the correlation of differentially expressedgenes that are associated with canonical pathways in INGENUITY pathwayanalysis. Total RNA were extracted from 56 pairs of fresh-frozen (FF)primary never smoker lung adenocarcinomas, and analyzed by WSB1 statususing INGENUITY pathway analysis.

FIGS. 3A and 3B are a series of tables listing gene expression profilesdetermined using total RNA from 56 pairs of FF primary never smoker lungadenocarcinomas, analyzed by WSB1 status using INGENUITY pathwayanalysis.

FIGS. 4A, 4B, and 4C are Kaplan-Meier graphs plotting survival ofpatients having metastatic human breast cancer (FIG. 4A), breast cancermetastasis to brain (FIG. 4B), and metastatic colon cancer (FIG. 4C),stratified according to high or low expression levels of WSB1(PROGgene).

FIG. 5 is a series of graphs plotting the correlation between WSB1 andHIF target gene expression in metastatic colon (top row) and urinarybladder (bottom row) cancer patients (GEO data set listed in Table 1).

FIGS. 6A-6D demonstrate that WSB1 upregulates HIF-1α activity. FIG. 6Ashows a series of representative immunoblots from experiments in whichHEK 293T cells were transfected with the indicated Myc, WSB1-Myc, andshRNA constructs, and levels of HIF-1α, Myc, WSB1, and β-actin wereexamined. FIG. 6B is a series of representative images of HIF-1α proteinexpression in HeLa cells that were transfected with WSB1 shRNA (#1) (GFPpositive) and then stained with DAPI (blue for DNA) and anti-HIF-1α(red). The position of a cell transfected by WSB1 shRNA is indicated bythe white arrowheads. The scale bar represents 10 μm. FIGS. 6C and 6Dcontain graphs plotting the results of quantitative polymerase chainreaction (qPCR) analyses of selected HIF-1α targets in HEK 293T cellsstably transfected with WSB1 cDNA (FIG. 6C) or WSB1 shRNA (FIG. 6D).Expression levels are relative to β-actin; data are normalized tocontrol cells. The results represent the means (±S.E.) of threeindependent experiments performed in triplicate. *P<0.05, **P<0.01,***P<0.001 versus control cells by one-way ANOVA.

FIGS. 7A and 7B demonstrate that HIF-1α levels and target geneexpression were decreased in cells depleted of WSB1. FIG. 7A containsrepresentative images of HIF-1α protein expression in HeLa cells thatwere transfected with WSB1 shRNA (#2) (GFP positive) and then stainedwith DAPI (blue for DNA) and anti-HIF-1α (red). The position of a celltransfected by WSB1 shRNA is indicated by the white arrowheads. Thescale bar represents 10 μm. FIG. 7B is a graph plotting relative mRNAlevels for the indicated genes in cells infected with WSB1 shRNA (#2)and then collected for qPCR analysis.

FIGS. 8A-8G demonstrate that WSB1 regulates pVHL ubiquitination anddegradation. FIG. 8A contains representative immunoblots fromco-immunoprecipitation (Co-IP) of endogenous HIF-1α and WSB1 fromextracts of HEK 293 T cells. FIG. 8B contains representative immunoblotsfrom Co-IP of endogenous pVHL and WSB1 from extracts of HEK 293T cells.FIG. 8C contains representative immunoblots showing regulation of pVHLand HIF-1α protein levels by WSB1 in a dose dependent manner. Cells weretransfected with the indicated amounts of WBS1 constructs, and pVHL andHIF-1α levels were examined. FIG. 8D contains representative immunoblotsshowing that down regulation of pVHL levels by WSB1 was reversed by MG132. Cells transfected with WSB1-Myc were left untreated or treated withMG 132, and pVHL levels were examined. FIG. 8E contains representativeimmunoblots showing pVHL levels in cells transfected with the indicatedWSB1 shRNAs. FIG. 8F contains representative immunoblots (left panel)and a graph (right panel), showing pVHL levels in cells stablyexpressing control shRNA, WSB1 shRNA, or WSB1 shRNA together withshRNA-resistant WSB1. Cells were treated with cycloheximide (0.1 mg/ml)and harvested at the indicated times. The graph quantifies the VHLprotein expression levels shown in the left panel. **P<0.01,***P<0.001versus control shRNA by one way ANOVA. FIG. 8G contains representativeimmunoblots for cells that were transfected with the indicatedconstructs and were then treated with MG 132. Ubiquitinated proteinswere pulled down under denaturing conditions by Ni-NTA Agarose andanalyzed by immunoblotting. pVHL-Ub_(n), polyubiquitinated pVHL.

FIGS. 9A and 9B demonstrate that WSB1 regulates pVHL ubiquitination anddegradation. FIG. 9A contains representative immunoblots from Co-IP ofexogenous HIF-1α, pVHL, and WSB1 from extracts of HEK 293T cells. FIG.9B contains representative immunoblots from five lung, six pancreatic,and four breast cancer cell lines, as well as normal MCF10A cells, thatwere analyzed by immunoblotting for the indicated proteins.

FIGS. 10A-10E demonstrate that WSB1 interacts with and degrades pVHLthrough its SOCS domain. FIG. 10A is a diagram of WT WSB1 andcorresponding deletion mutants (ΔWD 1-3, ΔWD 1-5, ΔSOCS, and Δ6-SOCS) asused in Co-IP experiments with pVHL. Plus and minus symbols indicate thebinding affinity of each WSB1 protein for pVHL. Arrows indicate theeffect of the WSB1 proteins (decreasing pVHL stability). FIG. 10Bcontains representative immunoblots for cells transfected with theindicated plasmids. For the blots in the left panel, cells werepretreated with MG 132, and the WSB1-pVHL interaction was examined byIP. In the right panel, pVHL levels were examined without MG 132pretreatment. FIG. 10C contains representative immunoblots showing thatdeleting the SOCS domain from WSB1 inhibits WSB1's E3 ligase activitytoward pVHL. Cells were transfected with the indicated constructs andthen treated with MG 132. Ubiquitinated proteins were pulled down underdenaturing conditions by Ni-NTA agarose and analyzed by immunoblotting.*non-specific band. FIG. 10D is a representative immunoblot for an invitro binding assay of recombinant WSB1 with pVHL. FIG. 10E containsrepresentative immunoblots for WSB1 (WT or ΔSOCS) that was purified fromcells and used for in vitro ubiquitination reactions with recombinantpVHL.

FIGS. 11A-11D demonstrate that WSB1 regulates pVHL under hypoxiaconditions. FIG. 11A contains representative immunoblots fromexperiments in which endogenous WSB1 was co-immunoprecipitated with pVHLfrom 293T cell extracts under hypoxia for the indicated time. The 0 timepoint represents lysates from normoxic cultures that were prepared atthe time of transfer to hypoxia. FIG. 11B contains representativeimmunoblots (left panel) and RT-PCR results (right panel) showingHIF-1α, HIF-2α, and WSB1 protein levels, as well as levels of HIF-1α,GULT1, WSB1 and VHL mRNA, after the indicated time of hypoxia. FIG. 11Cis a picture of a representative gel showing the ubiquitination of pVHLunder hypoxia conditions in HEK 293 cells transfected with control orWSB1 shRNA, as indicated. FIG. 11D contains representative immunoblotsshowing HIF-1α expression in cells transfected with control or WSB1shRNA and subjected to hypoxia conditions.

FIGS. 12A and 12B demonstrate that WSB1 interacts with pVHL in hypoxiamimic conditions. FIG. 12A contains representative immunoblots for cellstreated with CoCl₂ and then collected for immunoprecipitation (IP) andimmunoblot analysis. FIG. 12B contains representative immunoblotsshowing levels of HIF-1α, HIF-2α, pVHL, and WSB1 after CoCl₂ treatment.

FIGS. 13A-13D demonstrate that WSB1 promotes cancer cell invasion andmigration by inhibiting pVHL. FIG. 13A contains representativeimmunoblots showing pVHL and HIF-1α levels in cells transfected with theindicated constructs. FIG. 13B shows results from a trans-well invasionassay of RCC4 (left panel, top) or RCC4/VHL (left panel, bottom) cellsstably transfected with the indicated shRNAs or plasmids. The plot(right panel) shows the quantification of the area covered by theinvasion cells, relative to the control. Results represent the means(±S.E.) of three independent experiments performed in triplicate.*P<0.05, ***P<0.001 versus control cells by one-way ANOVA. FIG. 13Cshows the quantification of wound-healing assays of RCC4 or RCC4/VHLcell lines stably transfected with the indicated plasmids or shRNA. Theleft panel contains a graph plotting the means and s.d. of threeindependent experiments performed in triplicate. *, P<0.05, ***, P<0.001versus control cells by one-way ANOVA. The right panel containsrepresentative images of the assay. FIG. 13D shows the results fromexperiments in which RCC4 or RCC4/VHL cells were infected with theindicated constructs and HIF-1α activity was assayed by a HIF-1αluciferase reporter assay. The graph in the left panel shows the means(±S.E.) of three independent experiments performed in triplicate.*P<0.05, §§§§ P<0.0001 versus control cells by one-way ANOVA. The rightpanel contains representative immunoblots using the indicatedantibodies.

FIGS. 14A-14G demonstrate that WSB1 promotes cancer cell invasion,migration, and metastasis by enhancing HIFs. FIG. 14A containsrepresentative immunoblots for cells transduced with or without theindicated viral vectors. FIG. 14B is a graph plotting the results of atrans-well invasion assay for 786-O or 786-O/VHL cells stably expressingthe indicated shRNAs or plasmids. The graph shows quantification of thearea covered by the invasion cells, relative to the control. Resultsrepresent the means (±S.E.) of three independent experiments performedin triplicate. **P<0.01 versus vector virus injected cells; *P<0.05versus control shRNA infected cells by one-way ANOVA. FIG. 14C is agraph plotting the quantification of wound-healing assays using 786-O or786-O/VHL cell lines stably transfected with the indicated plasmids orshRNA. The mean and s.d. of one representative experiment, out of threeindependent experiments performed in triplicate, are shown (***P<0.001versus vector virus injected cells by one-way ANOVA). FIG. 14D containsrepresentative images from the experiments quantified in the graph ofFIG. 14C. For FIGS. 14E and 14F, lentiviral-driven shRNA was used todeplete endogenous WSB1 from MDA-MB-231 cells, which were then rescuedwith an RNAi-resistant WSB1 (Wt or ΔSOCS). FIG. 14E is a graph plottingHIF-1α activity in the cells. Results represent the means (±S.E.) ofthree independent experiments performed in triplicate. ***P<0.001 versuscontrol shRNA virus infected cells; ****P<0.0001 versus WSB1 shRNAinfected cells by one-way ANOVA. FIG. 14F contains representativeimmunoblots for cells transfected with the indicated constructs and thenimmunoblotted for the indicated proteins. FIG. 14G containsrepresentative images from wound healing experiments in whichlentiviral-driven shRNA was used to deplete endogenous WSB1 fromRCC4/VHL cells, which were then rescued with an RNAi-resistant WSB1 (Wtor ΔSOCS).

FIG. 15 demonstrates that WSB1 regulates HIF-1α in a HIPK2 independentmanner. Left panel: Cells were infected with the indicated shRNA andassayed by wound healing experiments. Right panel: Expression of theindicated proteins was examined by immunoblotting.

FIGS. 16A-16E demonstrate that WSB1 promotes cancer cell metastasis andis negatively correlated with pVHL in various human cancers. FIGS.16A-16C contain results from lung or liver colonization assays of micethat were intravenously injected with B16F10 cells stably infected withthe indicated viral construct or shRNA. In particular, FIG. 16A containsrepresentative images of from lung and liver, FIG. 16B contains a pairof graphs plotting the number of metastatic foci per lung (left) orliver (right) section, and FIG. 16C contains representative immunoblots.*P<0.05, **P<0.01, ***P<0.001 versus control cells by one-way ANOVA.FIG. 16D is a series of graphs plotting the quantity of pVHL-positivecells in slides from human patients with non-small cell lung cancer(NSCLC) adenocarcinoma, NSCLC squamous cell carcinoma, metastaticcolorectal cancer, and metastatic breast cancer, based on high or lowWSB1 expression. P values were calculated by Student's t-test. FIG. 16Eis a schematic model.

FIGS. 17A and 17B show immunohistochemical (IHC) staining of WSB1 andpVHL in human NSCLC tissue microarrays (TMA). FIG. 17A showsrepresentative immunostaining intensities: 0 (negative), 1+ (weak), 2+(moderate), and +3 (strong). FIG. 17B contains representative IHC imagesof WSB1 and pVHL in adenocarcinoma and squamous cell carcinoma, comparewith normal lung tissues. Serial tumor sections from the same patientwere processed.

DETAILED DESCRIPTION

This document is based, at least in part, on the discoveries that WSB1acts as an E3 ligase for pVHL, promotes HIF stabilization under both ofnormoxia and hypoxia conditions, is upregulated in human cancers andassociated with poor prognosis, and promotes cancer cell invasionmetastasis through the pVHL-HIF pathway. These discoveries are describedin further detail in the Examples below. Given these discoveries, thisdocument provides materials and methods that can be used to treat cancerand reduce metastasis by inhibiting the activity of WSB1, increasing theactivity of pVHL, or both.

WSB1 is a member of the WD-protein subfamily, containing severalWD-repeats spanning most of the protein and a SOCS box in the C-terminalregion. The human WSB1 protein has the amino acid sequence:

(SEQ ID NO: 1) MASFPPRVNEKEIVRLRTIGELLAPAAPFDKKCGRENWTVAFAPDGSYFAWSQGHRTVKLVPWSQCLQNFLLHGTKNVTNSSSLRLPRQNSDGGQKNKPREHIIDCGDIVWSLAFGSSVPEKQSRCVNIEWHRFRFGQDQLLLATGLNNGRIKIWDVYTGKLLLNLVDHTEVVRDLTFAPDGSLILVSASRDKTLRVWDLKDDGNMMKVLRGHQNWVYSCAFSPDSSMLCSVGASKAVFLWNMDKYTMIRKLEGHHHDVVACDFSPDGALLATASYDTRVYIWDPHNGDILMEFGHLFPPPTPIFAGGANDRWVRSVSFSHDGLHVASLADDKMVRFWRIDEDYPVQVAPLSNGLCCAFSTDGSVLAAGTHDGSVYFWATPRQVPSLQHLCRMSIRRVMPTQEVQELPIPSKLLEFLSYRI (NCBI Reference NP_056441),and is encoded by the nucleotide sequence:

(SEQ ID NO: 2) agatatctccggcgccgcccgccattttgactccagtgtctcgtttgcagtcggcgctttaggggaactgtcttcctccgcaggcgcgaggctgggtacagggtctattgtctgtggttgactccgtactttggtctgaggccttcgggagctttcccgaggcagttagcagaagccgcagcggccgcccccgcccgtctcctctgtccctgggcccgggagggaccaacttggcgtcacgcccctcagcggtcgccactctcttctctgttgttgggtccgcatcgtattcccggaatcagacggtgccccatagatggccagctttcccccgagggtcaacgagaaagagatcgtgagattacgtactataggtgaacttttagctcctgcagctccttttgacaagaaatgtggtcgtgaaaattggactgttgcttttgctccagatggttcatactttgcttggtcacaaggacatcgcacagtaaagcttgttccgtggtcccagtgccttcagaactttctcttgcatggcaccaagaatgttaccaattcaagcagtttaagattgccaagacaaaatagtgatggtggtcagaaaaataagcctcgtgaacatattatagactgtggagatatagtctggagtcttgcttttgggtcatcagttccagaaaaacagagtcgctgtgtaaatatagaatggcatcgcttcagatttggacaagatcagctacttcttgctacagggttgaacaatgggcgtatcaaaatatgggatgtatatacaggaaaactcctccttaacttggtagatcatactgaagtggtcagagatttaacttttgctccagatggaagcttgatcctggtgtcagcttcaagagacaaaactctcagagtatgggacctgaaagatgatggaaacatgatgaaagtattgagggggcatcagaattgggtgtacagctgtgcattctctcctgactcttctatgctgtgttcagtcggagccagtaaagcagttttcctttggaatatggataaatacaccatgatacggaaactagaaggacatcaccatgatgtggtagcttgtgacttttctcctgatggagcattactggctactgcatcttatgatactcgagtatatatctgggatccacataatggagacattctgatggaatttgggcacctgtttcccccacctactccaatatttgctggaggagcaaatgaccggtgggtacgatctgtatcttttagccatgatggactgcatgttgcaagccttgctgatgataaaatggtgaggttctggagaattgatgaggattatccagtgcaagttgcacctttgagcaatggtctttgctgtgccttctctactgatggcagtgttttagctgctgggacacatgacggaagtgtgtatttttgggccactccacggcaggtccctagcctgcaacatttatgtcgcatgtcaatccgaagagtgatgcccacccaagaagttcaggagctgccgattccttccaagcttttggagtttctctcgtatcgtatttagaagattctgccttccctagtagtagggactgacagaatacacttaacacaaacctcaagctttactgacttcaattatctgtttttaaagacgtagaagatttatttaatttgatatgttcttgtactgcattttgatcagttgagcttttaaaatattatttatagacaatagaagtatttctgaacatatcaaatataaatttttttaaagatctaactgtgaaaacatacatacctgtacatatttagatataagctgctatatgttgaatggacccttttgcttttctgatttttagttctgacatgtatatattgcttcagtagagccacaatatgtatctttgctgtaaagtgcaaggaaattttaaattctgggacactgagttagatggtaaatactgacttacgaaagttgaattgggtgaggcgggcaaatcacctgaggtcagcagtttgagactagcctggcaaacatgatgaaaccctgtctctactaaaaatacaaaaaaaaaaaaaattagccaggcgtggtggtgcacacctgtagtcctagctacttgggaggctgaggcaggagaattgcttgaacccaggaggtggaggttgcagtaagccaagatcacaccactgcactccaacctggacaacagagcgagactccatctcaaaaaaaaaaaaaaattgtgttgcctcatacgaaatgtatttggttttgttggagagtgtcagactgatctggaagtgaaacacagtttatgtacagggaaaaggattttattatccttaggaatgtcatccaagacgtagagcttgaatgtgacgttatttaaaaacaacaacaaagaaggcagagccaggatataactagaaaaaggatgtctttttttttttttttactccccctctaaacactgctgctgccttaattttagaaagcagcttactagtttaccatgtggtataaagtattataaattgttgtgaatttgaagaatccgtctactgtattattgctaaatattttgtttatactaagggacaattattttaagaccatggatttaaaaaaaaaaaaaaaaactctgtttctgcaggggatgatattggtgagttgccaaagaagcaatacagcatatctgatttgccttctgttgtttatcttacctgcagatattaagaatgtatgcattatgtaaaatgctcaattatatatttttgttgagttttttaattaaagacttgttaaaaaaaaaaaaaaaa (NCBI Reference NM_015626).

pVHL has ubiquitin ligase E3 activity, and is a component of a proteincomplex that includes elongin B, elongin C, and cullin-2. The complex isinvolved in the ubiquitination and degradation of hypoxia-induciblefactor (HIF), which is a transcription factor that plays a central rolein the regulation of gene expression by oxygen. The human pVHL proteinhas the amino acid sequence:

(SEQ ID NO: 3) MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD (NCBI Reference NP_000542.1),and is encoded by the nucleotide sequence:

(SEQ ID NO: 4) cctcgcctccgttacaacggcctacggtgctggaggatccttctgcgcacgcgcacagcctccggccggctatttccgcgagcgcgttccatcctctaccgagcgcgcgcgaagactacggaggtcgactcgggagcgcgcacgcagctccgccccgcgtccgacccgcggatcccgcggcgtccggcccgggtggtctggatcgcggagggaatgccccggagggcggagaactgggacgaggccgaggtaggcgcggaggaggcaggcgtcgaagagtacggccctgaagaagacggcggggaggagtcgggcgccgaggagtccggcccggaagagtccggcccggaggaactgggcgccgaggaggagatggaggccgggcggccgcggcccgtgctgcgctcggtgaactcgcgcgagccctcccaggtcatcttctgcaatcgcagtccgcgcgtcgtgctgcccgtatggctcaacttcgacggcgagccgcagccctacccaacgctgccgcctggcacgggccgccgcatccacagctaccgaggtcacctttggctcttcagagatgcagggacacacgatgggcttctggttaaccaaactgaattatttgtgccatctctcaatgttgacggacagcctatttttgccaatatcacactgccagtgtatactctgaaagagcgatgcctccaggttgtccggagcctagtcaagcctgagaattacaggagactggacatcgtcaggtcgctctacgaagatctggaagaccacccaaatgtgcagaaagacctggagcggctgacacaggagcgcattgcacatcaacggatgggagattgaagatttctgttgaaacttacactgtttcatctcagcttttgatggtactgatgagtcttgatctagatacaggactggttccttccttagtttcaaagtgtctcattctcagagtaaaataggcaccattgcttaaaagaaagttaactgacttcactaggcattgtgatgtttaggggcaaacatcacaaaatgtaatttaatgcctgcccattagagaagtatttatcaggagaaggtggtggcatttttgcttcctagtaagtcaggacagcttgtatgtaaggaggtttgtataagtaattcagtgggaattgcagcatatcgtttaattttaagaaggcattggcatctgcttttaatggatgtataatacatccattctacatccgtagcggttggtgacttgtctgcctcctgctttgggaagactgaggcatccgtgaggcagggacaagtctttctcctctttgagaccccagtgcctgcacatcatgagccttcagtcagggtttgtcagaggaacaaaccaggggacactttgttagaaagtgcttagaggttctgcctctatttttgttggggggtgggagaggggaccttaaaatgtgtacagtgaacaaatgtcttaaagggaatcatttttgtaggaagcattttttataattttctaagtcgtgcactttctcggtccactcttgttgaagtgctgttttattactgtttctaaactaggattgacattctacagttgtgataatagcatttttgtaacttgccatccgcacagaaaatacgagaaaatctgcatgtttgattatagtattaatggacaaataagtttttgctaaatgtgagtatttctgttcctttttgtaaatatgtgacattcctgattgatttgggtttttttgttgttgttgttttgttttgttttgtttttttgagatggagtctcactcttgtcacccaggctggagtgcagtggcgccatctcggctcactgcaacctctgcctcctgggttcacgtaatcctcctgagtagctgggattacaggcgcctgccaccacgctggccaatttttgtacttttagtagagacagtgtttcgccatgttggccaggctggtttcaaactcctgacctcaggtgatccgcccacctcagcctcccaaaatggtgggattacaggtgtgtgggccaccgtgcctggctgattcagcattttttatcaggcaggaccaggtggcacttccacctccagcctctggtcctaccaatggattcatggagtagcctggactgtttcatagttttctaaatgtacaaattcttataggctagacttagattcattaactcaaattcaatgcttctatcagactcagttttttgtaactaatagatttttttttccacttttgttctactccttccctaatagctttttaaaaaaatctccccagtagagaaacatttggaaaagacagaaaactaaaaaggaagaaaaaagatccctattagatacacttcttaaatacaatcacattaacattttgagctatttccttccagcctttttagggcagattttggttggtttttacatagttgagattgtactgttcatacagttttataccctttttcatttaactttataacttaaatattgctctatgttagtataagcttttcacaaacattagtatagtctcccttttataattaatgtttgtgggtatttcttggcatgcatctttaattccttatcctagcctttgggcacaattcctgtgctcaaaaatgagagtgacggctggcatggtggctcccgcctgtaatcccagtactttggaaagccaaggtaagaggattgcttgagcccagaacttcaagatgagcctgggctcatagtgagaacccatctatacaaaaaatttttaaaaattagcatggcggcacacatctgtaatcctagctacttggcaggctgaggtgagaagatcattggagtttaggaattggaggctgcagtgagccatgagtatgccactgcactccagcctgggggacagagcaagaccctgcctcaaaaaaaaaaaaaaaaaaaaaatcaggccgggcatggtggctcacgcctgtaatcccagcactttgggaggtcgaggtgggcagatcacctgaggtcaggagttcgagaccagcctggccaacatggtaaaaccccatttctactaaaaaatacaagaattagctgggtgtggtggcgcatgcctgtaatcctagctactcaggaggctgaggcaggagaatcacttgaacccaggaggcgaagattgcagtgagctgatatcgcaccattgtactccagcctgtgtgacagagcaatactatgtctcaaaaaaaaaaaaaaattcaaatcagagtgaagtgaatgagacactccagttttccttctactccgaatttcaactgattttagctcctcattcaacattcaacaaatagtcttttttttttttttttttatttttttttgagatggagtctcactctgttgcccaggctggagtgcagtggtgcgatctctgctcactacaagctctgcctcccgagttcaagtgattctcctggctcaccctcctgagtagctgggattacaggcgcctgccaccatgcctggctaattttgtgtttttagtggagacggggtttcaccatgttgtccaggatggtcttgatctcctgaccttgtgatccacccacctcagcctcccaaagtgctgggattacaggtgtgagccaccgcgtccagccagattattattttttttaagctgtattgtgtcaaaatgatagttcatgctcctatgttaaaacctgcaggccgagcacagtggctcatgcctgtaatcccagcattttgggagaccaaggcggatggatcacctgaggtcaggagctgaagaccagcctggctaacatggtgaaacctcatctccacttaaaatacaaaaattgccggccgcggcggctcatgcctgtaatcccagcactttgggaggcctaggcgggtggatcacgaggtcaggaaatcgagaccatcctggctaacacgggtgaaaccccgtctctattaaaaaatagaaaaaattaggcgggcgtggtggtgagcgcctgtagtcccagctactcgagagcctgaggcaggagaatggcatgaacctggaaggcggagcttgcagtgagctgagatggtgccactgcactctaacctgggcgacagagtgagacaccgtctcaaaaaaaaaaacaaaaaacaaaaattatccaggtgtggcggtgggcgcctgtgaggcaggcgaatctcttgaacccgggaggcggaggttgcagtgagccaagatcacaccattgcactccagcctgggcaacaagagtgaaattccatctcaaaaagaaaccaaaaaaacaaaaaaaaaacatgccgtttgagtactgtgtttttggtgttgtccaaggaaaattaaaaacctgtagcatgaataatgtttgtttttcatttcgaatcttgtgaatgtattaaatatatcgctcttaagagacggtgaagttcctatttcaagtttttttttttttttttttttttaaagctgttttttaatacattaaatggtgctgagtaaaggaaatag (NCBI Reference NM_000551).

In some embodiments, this document provides agents that can reduce theactivity of WSB1. An agent can be targeted to WSB1 to inhibit itsactivity by, for example, reducing expression of WSB1 protein, or byreducing the activity of WSB1 protein that is expressed. Examples ofsuch agents may include small molecules and inhibitory nucleic acids(e.g., siRNA, miRNA, shRNA molecules, and antisense oligonucleotides).For example, small molecules targeting the WD40 and/or SOCS domain ofWSB1 may be particularly useful. Antagonistic antibodies that bind toWSB1 also may be useful as agents for reducing WSB1 activity. Methodsfor obtaining and testing the activities of such agents include thoseknown in the art.

In some embodiments, this document provides agents that can increase theactivity of pVHL. An agent can be targeted to pVHL to increase itsactivity by, for example, increasing the expression of pVHL protein, orby increasing the activity or stability of pVHL protein that isexpressed. Examples of such agents may include small molecules, nucleicacids encoding pVHL, and pVHL polypeptides themselves. Agonisticantibodies that bind to pVHL also may be useful as agents for increasingpVHL activity. Again, methods for obtaining and testing the activitiesof such agents include those known in the art.

For example, methods of screening for small molecule inhibitors oractivators (antagonists or agonists) of polypeptides such as WSB1 orpVHL1 are known in the art. In some embodiments, for example, cells canbe cultured with one or more candidate small molecules, and the effecton WSB1 or pVHL expression, stability, or activity can be assessed usingtechniques such as northern blotting, Western blotting, or otherimmunological methods.

Methods for designing and making inhibitory nucleic acids also are knownin the art. Such agents can reduces the level of mRNA that encodes aWSB1 polypeptide. For example, a WSB1 antagonist can be an agent thatreduces transcription of nucleic acid encoding a WSB1 polypeptide, orpromotes degradation of mRNA encoding a WSB1 polypeptide (e.g., by RNAinterference (RNAi)), or inhibits posttranscriptional processing (e.g.,splicing or nuclear export) of mRNA encoding a WSB1 polypeptide. Such anagent can inhibit protein synthesis from WSB1 mRNA (e.g., by RNAi), orpromote degradation of WSB1 protein, thereby reducing the level of WSB1polypeptide in a subject. Small interfering RNA (siRNA) molecules can besynthesized in vitro or made from a DNA vector in vivo. In some cases, asiRNA molecule can contain a backbone modification to increase itsresistance to serum nucleases and increase its half-life in thecirculation. Such modification can be made as described elsewhere (Chiuet al., RNA 2003, 9:1034-1048; and Czauderna et al., Nucleic Acids Res2003, 31:2705-2716). In some cases, a small hairpin RNA (shRNA, whichcan be converted to a siRNA) can be used as a WSB1 antagonist.

Methods for making antibodies having specific binding affinity for aWSB1 or pVHL polypeptide can include recombinant production of thepolypeptide, purification of the polypeptide from a biological sample(e.g., a heterologous expression system), or chemical synthesis of thepolypeptide. For example, a polypeptide having the amino acid sequenceset forth in SEQ ID NO:1 or SEQ ID NO:3, or a fragment thereof that isat least six amino acids in length, can be used to immunize an animal.Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin anddinitrophenol. Monoclonal antibodies can be prepared using a polypeptideprovided herein and standard hybridoma technology. In particular,monoclonal antibodies can be obtained by any technique that provides forthe production of antibody molecules by continuous cell lines in culturesuch as described by Kohler et al., Nature, 256:495 (1975), the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72(1983); Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026 (1983)), andthe EBV-hybridoma technique (Cole et al., “Monoclonal Antibodies andCancer Therapy,” Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodiescan be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD,and any subclass thereof. The hybridoma producing the monoclonalantibodies can be cultivated in vitro and in vivo.

This document also provides methods and materials related to identifyingagonists or antagonists of WSB1-mediated ubiquitination of pVHL. Forexample, this document provides methods and materials for using WSB1polypeptides and pVHL polypeptides (e.g., ubiquitinated pVHLpolypeptides) to identify agents that increase or decrease the effectsof the WSB1 polypeptides on the pVHL polypeptides. In some cases, thestability of ubiquitinated pVHL polypeptides treated with WSB1polypeptides in the presence and absence of a test agent can be assessedto determine whether or not the test agent increases or decreases thestability of the ubiquitinated pVHL polypeptides. An agent thatincreases the stability of the ubiquitinated pVHL polypeptides in amanner dependent on the WSB1 polypeptide can be an antagonist ofWSB1-mediated action on pVHL polypeptides, and an agent that decreasesthe stability of the ubiquitinated pVHL polypeptides in a mannerdependent on the WSB1 polypeptide can be an agonist of WSB1-mediatedaction on pVHL polypeptides. The stability of ubiquitinated pVHLpolypeptides can be assessed using polypeptide assays capable ofdetecting intact full-length polypeptide or degraded polypeptides, forexample. Agonists and antagonists also can be identified by screeningtest agents (e.g., from synthetic compound libraries and/or naturalproduct libraries). Test agents can be obtained from any commercialsource and can be chemically synthesized using methods that are known tothose of skill in the art. Test agents can be screened and characterizedusing in vitro cell-based assays, cell free assays, and/or in vivoanimal models.

Methods of making nucleic acids, including inhibitory nucleic acids, areknown in the art. As used herein, the term “nucleic acid” refers to bothRNA and DNA, including mRNA, cDNA, genomic DNA, synthetic (e.g.,chemically synthesized) DNA, and nucleic acid analogs. A nucleic acidcan be double-stranded or single-stranded, and where single-stranded,can be the sense strand or the antisense strand. In addition, nucleicacid can be circular or linear. Nucleic acid analogs can be modified atthe base moiety, sugar moiety, or phosphate backbone to improve, forexample, stability, hybridization, or solubility of a nucleic acid.Modifications at the base moiety include deoxyuridine fordeoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety can include modification of the 2′ hydroxyl of the ribose sugarto form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered, morpholino ring, orpeptide nucleic acids, in which the deoxyphosphate backbone is replacedby a pseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, Antisense Nucleic Acid Drug Dev7:187-195, 1997; and Hyrup et al. Bioorgan Med Chem 4:5-23, 1996. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

An isolated nucleic acid as provided herein can comprise or consist of asequence that encodes the amino acid sequence set forth in SEQ ID NO:3,for example, or a portion thereof. In some embodiments, such a nucleicacid can contain the human nucleic acid sequence set forth in SEQ IDNO:4.

Typically, an isolated nucleic acid is at least 10 nucleotides in length(e.g., 10 to 100, 15 to 150, 20 to 200, 25 to 250, 30 to 300, 40 to 400,50 to 500, 75 to 750, 100 to 1000, or more than 1000 nucleotides inlength). Nucleic acid molecules that are less than full-length can beuseful, for example, as primers or probes for diagnostic purposes.Isolated nucleic acid molecules can be produced by standard techniques,including, without limitation, common molecular cloning and chemicalnucleic acid synthesis techniques. For example, polymerase chainreaction (PCR) techniques can be used. PCR refers to a procedure ortechnique in which target nucleic acids are enzymatically amplified.Sequence information from the ends of the region of interest or beyondtypically is employed to design oligonucleotide primers that areidentical in sequence to opposite strands of the template to beamplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 15 to 50 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length. Forexample, a primer can be 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, or 45 nucleotides in length. A primer can bepurified from a restriction digest by conventional methods, or can bechemically synthesized. Primers typically are single-stranded formaximum efficiency in amplification, but a primer can bedouble-stranded. Double-stranded primers are first denatured (e.g.,treated with heat) to separate the strands before use in amplification.General PCR techniques are described, for example in PCR Primer: ALaboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring HarborLaboratory Press, 1995. When using RNA as a source of template, reversetranscriptase can be used to synthesize a complementary DNA (cDNA)strand. Ligase chain reaction, strand displacement amplification,self-sustained sequence replication or nucleic acid sequence-basedamplification also can be used to obtain isolated nucleic acids asdescribed elsewhere (Lewis, Genetic Engineering News 12(9):1, 1992;Guatelli et al., Proc Natl Acad Sci USA 87:1874-1878, 1990; and Weiss,Science 254:1292, 1991).

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

As used herein, a “vector” is a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment can be inserted so as to bringabout the replication of the inserted segment. A vector can be anexpression vector. An “expression vector” is a vector that includes oneor more expression control sequences, and an “expression controlsequence” is a DNA sequence that controls and regulates thetranscription and/or translation of another DNA sequence.

In an expression vector provided herein, the nucleic acid can beoperably linked to one or more expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. Examples of expression control sequencesinclude promoters, enhancers, and transcription terminating regions. Apromoter is an expression control sequence composed of a region of a DNAmolecule, typically within 100 nucleotides upstream of the point atwhich transcription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it can be necessary to position the translation initiationsite of the translational reading frame of the polypeptide between oneand about fifty nucleotides downstream of the promoter. Enhancersprovide expression specificity in terms of time, location, and level.Unlike promoters, enhancers can function when located at variousdistances from the transcription site. An enhancer also can be locateddownstream from the transcription initiation site. A coding sequence is“operably linked” and “under the control” of expression controlsequences in a cell when RNA polymerase is able to transcribe the codingsequence into mRNA, which then can be translated into the polypeptideencoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,poxviruses, adenoviruses, and adeno-associated viruses. Numerous vectorsand expression systems are commercially available from such corporationsas Novagen (Madison, Wis.), Clontech Laboratories (Mountain View,Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or FLAG™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

This document also provides host cells containing a nucleic acidmolecule and/or nucleic acid vector as described herein. The term “hostcells” refers to prokaryotic cells and eukaryotic cells into which anucleic acid molecule or vector can be introduced. Any method can beused to introduce nucleic acid into a cell. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer can be usedintroduce nucleic acid into cells. In addition, naked DNA can bedelivered directly to cells in vivo as described elsewhere (U.S. Pat.Nos. 5,580,859 and 5,589,466).

Interfering RNA molecules can be effective in suppressing accumulationof mRNAs and the polypeptides that they encode. A “small interferingRNA” or “short interfering RNA” (siRNA) or “short hairpin RNA” (shRNA)is a double-stranded RNA molecule that is complementary to a targetnucleic acid sequence. A double-stranded RNA molecule can be formed bycomplementary pairing between a first RNA portion and a second RNAportion. The length of each portion generally is less than 30nucleotides in length (e.g., 25 to 30, 20 to 25, 15 to 20, or 10 to 15nucleotides). In some embodiments, for example, the length of eachportion can be 19 to 25 nucleotides. In some siRNA molecules, thecomplementary first and second portions of the RNA molecule are the“stem” of a hairpin structure. The two portions can be joined by alinking sequence, which can form a “loop” in the hairpin structure. Thelinking sequence can vary in length. In some embodiments, the linkingsequence can be 5 to 8, 6 to 9, 7 to 10, 8 to 11, 9 to 12, or 10 to 13nucleotides in length. The first and second portions are complementarybut may not be completely symmetrical, as the hairpin structure maycontain 3′ or 5′ overhang nucleotides (e.g., a 1, 2, 3, 4, or 5nucleotide overhang).

Methods for identifying target sequences and generating interfering RNAstargeted to those sequences include techniques known in the art, forexample. siRNA-mediated suppression of nucleic acid expression isspecific, as even a single base pair mismatch between siRNA and thetargeted nucleic acid can abolish the action of RNA interference. It isnoted that certain embodiments of inhibitory nucleic acids against WSB1are described in the Examples below.

One or more agents that modulate the levels or activity of WSB1 and/orpVHL can be incorporated into a composition for administration to amammal (e.g., a research animal or a human patient diagnosed as havingcancer). For example, an agent or molecule that leads to reduced WSB1levels or increased pVHL levels as described herein can be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecular structures, or mixtures of compounds such as, for example,liposomes, receptor or cell targeted molecules, or oral, topical orother formulations for assisting in uptake, distribution and/orabsorption.

In some cases, a composition can contain one or more agents targeted toWSB1 or pVHL in combination with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include, for example,pharmaceutically acceptable solvents, suspending agents, or any otherpharmacologically inert vehicles for delivering compounds to a subject.Pharmaceutically acceptable carriers can be liquid or solid, and can beselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, and other pertinent transportand chemical properties, when combined with one or more therapeuticcompounds and any other components of a given pharmaceuticalcomposition. Exemplary pharmaceutically acceptable carriers include,without limitation: water, saline solution, binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g.,lactose or dextrose and other sugars, gelatin, or calcium sulfate),lubricants (e.g., starch, polyethylene glycol, or sodium acetate),disintegrates (e.g., starch or sodium starch glycolate), and wettingagents (e.g., sodium lauryl sulfate). In some embodiments, for example,an agent that modulates the level or activity of WSB1 and/or pVHL can becombined with a physiological salt solution such as 0.9% sodiumchloride, or an isotonic aqueous solution of sodium phosphate bufferedto a pH of 7.4.

The methods provided herein can include administering to a mammal (e.g.,a human or a non-human mammal) an agent that modulates the level oractivity of WSB1 and/or pVHL (e.g., an agent that decreases the level oractivity of WSB1, or that increases the level or activity of pVHL),under conditions such that the administration is therapeuticallyeffective against the development or progression of cancer. As usedherein, “therapeutically effective” refers to a reduction in one or moresymptoms of cancer, or a reduction in or prevention of cancerprogression (e.g., metastasis). Compositions containing one or moreagents as described herein can be given once or more daily, weekly,monthly, or even less often, or can be administered continuously for aperiod of time (e.g., hours, days, or weeks). In some cases,preparations can be designed to stabilize such agents and maintaineffective activity in a mammal for several days.

Pharmaceutical compositions containing one or more agents that modulateWSB1 and/or pVHL as described herein can be administered by a number ofmethods, depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration of the agents andcompositions provided herein can be, for example, oral or parenteral(e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations). Fortreating tissues in the central nervous system, an agent or compositioncan be administered by injection or infusion into the cerebrospinalfluid, typically with one or more agents capable of promotingpenetration of the polypeptides across the blood-brain barrier.

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders. Compositions and formulations forparenteral, intrathecal or intraventricular administration can includesterile aqueous solutions, which also can contain buffers, diluents andother suitable additives (e.g., penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers).

Pharmaceutical compositions include, without limitation, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other; in general, emulsions are either of thewater-in-oil (w/o) or oil-in-water (o/w) variety. Emulsion formulationshave been widely used for oral delivery of therapeutics due to theirease of formulation and efficacy of solubilization, absorption, andbioavailability.

Liposomes are vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that can contain the composition to bedelivered. Liposomes can be particularly useful due to their specificityand the duration of action they offer from the standpoint of drugdelivery. Liposome compositions can be formed, for example, fromphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Numerous lipophilic agents are commerciallyavailable, including LIPOFECTIN® (Invitrogen/Life Technologies,Carlsbad, Calif.) and EFFECTENE™ (Qiagen, Valencia, Calif.).

The agents and compositions useful in the methods provided herein canfurther encompass any pharmaceutically acceptable salts, esters, orsalts of such esters, or any other compound which, upon administrationto an animal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, this document provides pharmaceuticallyacceptable salts of agents that modulate the levels or activity of WSB1and/or pVHL, prodrugs and pharmaceutically acceptable salts of suchprodrugs, and other bioequivalents. The term “prodrug” indicates atherapeutic agent that is prepared in an inactive form and is convertedto an active form (i.e., drug) within the body or cells thereof by theaction of endogenous enzymes or other chemicals and/or conditions. Theterm “pharmaceutically acceptable salts” refers to physiologically andpharmaceutically acceptable salts of the agents provided herein (i.e.,salts that retain the desired biological activity of the agent withoutimparting undesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, but are not limited to, salts formed withcations (e.g., sodium, potassium, calcium, or polyamines such asspermine); acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ornitric acid); and salts formed with organic acids (e.g., acetic acid,citric acid, oxalic acid, palmitic acid, or fumaric acid).

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositionsprovided herein, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers.Furthermore, the composition can be mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,and aromatic substances. When added, however, such materials should notunduly interfere with the biological activities of the WSB1- and/orpVHL-modulating agents within the compositions provided herein. Theformulations can be sterilized if desired.

The pharmaceutical formulations, which can be presented conveniently inunit dosage form, can be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thedesired pharmaceutical carrier(s) or excipient(s). Typically, theformulations can be prepared by uniformly and bringing the activeingredients into intimate association with liquid carriers or finelydivided solid carriers or both, and then, if necessary, shaping theproduct. Formulations can be sterilized if desired, provided that themethod of sterilization does not interfere with the effectiveness of thepolypeptide contained in the formulation.

The compositions described herein can be formulated into any of manypossible dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. The compositionsalso can be formulated as suspensions in aqueous, non-aqueous or mixedmedia. Aqueous suspensions further can contain substances that increasethe viscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol, and/or dextran. Suspensions also cancontain stabilizers.

The agents and compositions described herein also can be combined withpackaging material and sold as kits for treating cancer. Components andmethods for producing articles of manufacture are well known. Thearticles of manufacture may combine one or more of the agents and/ormolecules provided herein. In addition, the article of manufacturefurther may include, for example, buffers or other control reagents forreducing or monitoring the symptoms of the cancer to be treated.Instructions describing how the agents and compositions are effectivefor reducing such symptoms can be included in such kits.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Materials and Methods

Cells, Cell Lines and Reagents:

All cell lines were sourced from commercial vendors. Human embryonickidney (HEK) 293T, HEK 293, HeLa cervix carcinoma cells, RCC4, RCC4/VHL,786-O, and 786-O/VHL human renal carcinoma cells were cultured inDulbecco's modified Eagle's media (DMEM, Gibco-Invitrogen). Five humanlung cancer lines (three adenocarcinoma—H522, H1650 and A549 and twolarge cell carcinoma—H460 and H1299), six human pancreatic cancer celllines (BxPC3, Hup-T3, Mia-Paca, Panc1, Pan 04.03, and ASPC1), four humanbreast cancer cell lines (HT-29, HCC1937, HCC1806, and MDA231), andmouse melanoma B16F10 cells were maintained in Eagle's Minimal EssentialMedia (EMEM) or RPMI 1640 (Gibco-Invitrogen, Grand Island, N.Y.). Allmedia contained 10% heat-inactivated FBS (Gibco-Invitrogen), sodiumbicarbonate (2 mg/ml; Sigma-Aldrich, St Louis, Mo.), penicillin (100units/ml), and streptomycin (100 μg/ml; Gibco-Invitrogen).N-carbobenzoxy-1-leucinylleucinyl-1-norleucinal (MG 132), cycloheximide(CHX), and CoCl₂ were purchased from Sigma-Aldrich.

In Vitro Assays:

After transfection with Myc or Myc-WSB1 (WT, ΔSOCS), cells werecollected for immunoprecipitation and/or immunoblot analysis. Afterimmunoprecipitation with Myc antibody for WSB1, samples were incubatedwith reaction buffer (50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl₂, 0.05%Nonidet P-40, and 0.5 mM dithiothreitol), Flag- or Myc-ubiquitin (5 mM),ATP (2 mM), and substrates (e.g., recombinant VHL) at 32° C. for 90minutes.

In Vivo Ubiquitination Assays:

For in vivo ubiquitination, cells were transfected with ubiquitin-Hisplasmid together with Myc, Myc-WSB1, or Myc-ΔSOCS, followed by treatmentwith MG 132 (10 μM). 48 hours post-transfection, cells were lysed withurea lysis buffer (8 M urea, 0.1 M Na₂HPO₄, 0.1 M Tris/HCl (pH 8.0),0.05% Tween 20, and 0.01 M imidazole). After centrifugation, thesupernatants were collected and incubated with 20 mL Ni-NTA agarosebeads (Qiagen) for four hours at 4° C. The precipitates were washedthree times with urea wash buffer (8 M urea, 0.1 M Na₂HPO₄, 0.1 MTris/HCl (pH 8.0), 0.05% Tween 20, and 0.02 M imidazole) and native washbuffer (0.1 M Na₂HPO₄, 0.1 M Tris/HCl (pH 8.0), 0.05% Tween 20, and 0.02M imidazole), and were boiled with SDS loading buffer and then subjectedto SDS-PAGE followed by immunoblot analysis.

In Vitro Binding Assay:

GST fusion proteins were prepared following standard protocols. For invitro binding assays, WSB1-(WT, ΔSOCS) GST fusion proteins bound to GSHSepharose were incubated with cell lysates. After washing, the boundproteins were separated by SDS-PAGE and immunoblotted with antibodies asdesired.

Statistical Analysis:

Each assay was performed in triplicate and independently repeated atleast three times. Results are presented as mean±standard error of mean(SEM). Statistical analyses were performed using GraphPad Prism software(version 4.02; GraphPad Software, San Diego, Calif.). One-way analysisof variance (ANOVA) followed by T-test was used to compare the results.A difference was considered significant if P<0.05. Statisticalsignificance was defined as P<0.05 (*), P<0.01(**), and P<0.001(***).

Gene Expression Profiling from FF Primary Never Smoker LungAdenocarcinoma Patients:

Gene expression profiling was described elsewhere (Jang et al., ClinCancer Res 18:3658-3667, 2012). Briefly, after RNA extraction and geneexpression profiling by microarray from 56 primary lung tumors, the datafor mRNA was processed and normalized through BeadStudio software,version 3.0. (Illumina Inc.) using the quantile normalization method,and then log 2 transformed and analyzed using the Partek Genomics Suite(Partek Inc.). To identify differentially expressed genes, the one-wayANOVA model was applied for all analyses. mRNAs with a fold change>±1.5at raw P value<0.01 and false discovery rate (FDR)<5% were consideredsignificant. FF tumors were further subclassified into WSB1 “high (H)”and “low (L)” groups based on the means of expression afternormalization, and mRNA expression was compared between WSB1-H andWSB1-L tumors. 2,534 mRNAs were found to be differentially expressedbased on fold change>1.5 and FDR<5% using this analysis. These 2,534genes were analyzed using Ingenuity Pathway Analysis (IPA) 8.5 software(Ingenuity Systems, CA) to identify their biological functions in lungadenocarcinoma potentially modified by WSB1 expression level.

Plasmids:

Myc-tagged WSB1 (empty and WT, ΔWD1-2, ΔWD1-3, ΔN, ΔSOCS, and ΔC) wereprovided by Dr. Cheol Yong Choi (Sungkyunkwan University, Korea).HA-tagged HIF-1α and HA-tagged VHL were obtained from Addgene.

Transient Transfection and Stable Transduction:

shRNAs were infected using LIPOFECTAMINE® 2000 reagent (Invitrogen).Human WSB1, mouse WSB1, HIF-1α, and HIPK2 were obtained fromSigma-Aldrich and Open Biosystems.

WSB1 shRNA (Human):

(SEQ ID NO: 5) 5′-TGCTGTTGACAGTGAGCGCGGAGTTTCTCTCGTATCGTATTAGTGAAGCCACAGATGTAATACGATACGAGAGAAACTCCATGCCTACTGCCTCG GA-3′ (SEQ ID NO: 6)5′-TGCTGTTGACAGTGAGCGCGCTGTAAAGTGCAAGGAAATTTAGTGAAGCCACAGATGTAAATTTCCTTGCACTTTACAGCATGCCTACTGCCTC GGA-3′

WSB1 shRNA (Mouse):

(SEQ ID NO: 7) 5′-ACATGAGCTGCTGCTATATAT-3′ (SEQ ID NO: 8)5′-GCTTACTCCTTGTATCAGCTT-3′ HIF-1a shRNA (mouse) (SEQ ID NO: 9)5′-GTGATGAAAGAATTACCGAAT-3′ (SEQ ID NO: 10) 5′-TGCTCTTTGTGGTTGGATCTA-3′HIPK2 shRNA (mouse) (SEQ ID NO: 11)5′-GCTGTTGACAGTGAGCGACGAGTCAGTATCCAGCCCAATTAGTGAAGCCACAGATGTAATTGGGCTGGATACTGACTCGGTGCCTACTGCCTCGG A-3′ (SEQ ID NO: 12)5′-GCTGTTGACAGTGAGCGAGGAGAGTGCCGATGACTATAATAGTGAAGCCACAGATGTATTATAGTCATCGGCACTCTCCGTGCCTACTGCCTCGG A-3′

For transient overexpression studies, DNA plasmids were transfectedusing the LIPOFECTAMINE® 2000 reagent (Invitrogen). Stableoverexpression and silencing were obtained by transducing MDAMB-231, HEK293T, HEK 293, RCC4, RCC4/VHL, 786-O, 786-O/VHL, and H1299 cells withretroviral or lentiviral vectors. The efficiency of knockdown oroverexpression was assessed by Western blotting.

Migration and Invasion Assays:

For migration assays (wound healing assays), cells were seeded in 6-wellplates at a density of 15%/well and grown until confluence (around 3days). Complete medium was replaced with serum-free medium and cellswere incubated for 24 hours. Confluent cells (monolayer) were scrapedwith a P200 tip in each well (3 lines/well), and the medium was replacedwith complete medium. After 24 to 48 hours, the cells were fixed with3.7% paraformaldehyde (PFA). Photographs were obtained at 0, 1, and 2days. Cell migration was quantified by counting inside the scratch (attime 0 as standard) in each different fields of the wound.

For matrigel invasion assays, RCC4 or RCC4/VHL and 786-O or 786-O/VHLcells were infected with shRNAs in 10 cm dishes. After 8 hours, themedium was changed to serum-free medium and cells were grown to ˜80%confluence. Cells were seeded in 24-well invasion chambers (Corning,354480). Each sample was plated in triplicate (500,000 cells/insert).All protocols were followed as recommended standard protocols. Tomeasure cell invasion, filter were stained with 0.2% Crystal Violet andinvasion cells were counted.

Coimmunoprecipitation Assays, Immunoblotting and Antibodies:

To study endogenous WSB1/pVHL binding, cells were treated with orwithout 10 μM MG 132 (Sigma) for 2 hours. Cells were lysed by sonicationin NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%Nonidet P-40) containing 50 mM b-glycerophosphate, 10 mM NaF, and 1mg/ml each of pepstatin A and aprotinin, freshly supplemented withprotease inhibitor cocktail (Roche). Prior to immunoprecipitation,protein A-bound Agarose beads were incubated overnight with pVHLantibody (Cell Signaling, #2738), WSB1 antibody (Abcam, ab68953; Sigma,HPA003293; Proteintech, 1166-1-AP), or HIF-1α antibody (Abcam, ab51608)in PBS with 5% BSA at 4° C. ab68953 was used for most experiments(HPA003293 for IHC). Extracts were added before immunoprecipitation withprotein-A agarose at 4° C. for 4 hours. After three washings in bindingbuffer, co-purified proteins were analyzed by Western blotting.

For ubiquitination assays, HEK 293T cells were infected with theindicated shRNAs with HA-ubiquitin. Before harvesting, cells weretreated for 4 hours with proteasome inhibitor (10 μM MG 132; Sigma).Ubiquitination assays were then performed as described previously (Yuanet al., Cell 140:384-396, 2010).

To remove the heavy chain, heavy or light-chain-specific anti-mouse andanti-rabbit IgG secondary antibodies were obtained from JacksonImmunoresearch. Rabbit polyclonal antibodies recognizing HIF-2α werepurchased from Novus (NB100-122).

Immunofluorescence:

For immunofluorescence staining, HeLa cells were plated on glass coverslips and transfected with the indicated constructs. Cells were thenfixed in 3.7% paraformaldehyde for 10 minutes at room temperature, andstained using standard protocols. Immunofluorescence images were takenusing fluorescent microscopy (Nikon Microscope, Melville, N.Y.).

Real Time PCR or Reverse Transcription (RT)-PCR of cDNA:

Preparation of RNA and cDNA, as well as qRT-PCR, were performed asdescribed elsewhere (Lee et al., J Cell Sci 124:1911-1924, 2011). Thefollowing primers were used:

HIF-1α Forward:  (SEQ ID NO: 13) 5′-CATGGAAGGTATTGCACTGC-3′ Reverse: (SEQ ID NO: 14) 5′-CACACATACAATGCACTGTGG-3′ VEGFA Forward: (SEQ ID NO: 15) 5′-CCTTGCCTTGCTGCTCTACCTC-3′ Reverse:  (SEQ ID NO: 16)5′-TTCTGCCCTCCTCCTTCTGC-3′ CA90 Forward:  (SEQ ID NO: 17)5′-CAATATGAGGGGTCTCTGACTACAC-3′ Reverse:  (SEQ ID NO: 18)5′-GGAATTCAGCTGGACTGGCTCAGC-3′ ALDOC Forward:  (SEQ ID NO: 19)5′-GCGCTGTGTGCTGAAAATCAG-3′ Reverse:  (SEQ ID NO: 20)5′-CCACAATAGGCACAATGCCATT-3′ SAP30 Forward:  (SEQ ID NO: 21)5′-AGTTGGTTGCCACTTTAGGTC-3′ Reverse:  (SEQ ID NO: 22)5′-CCACGTCTCCTAGTGAACACC-3′ GULT1 Forward:  (SEQ ID NO: 23)5′-TCATCGTGGCTGAACTCTTCAG-3′ Reverse:  (SEQ ID NO: 24)5′-TCACACTTGGGAATCAGCCCC-3′

β-actin sequence were as described elsewhere (Lee et al., supra).

Cancer Data Collection and Processing:

Cancer datasets were obtained from Gene Expression Omnibus (online atncbi.nlm.nih.gov/geo) and PROGgene (online atwatson.compbio.iupui.edu/chirayu/proggene/database/?url=proggene),containing patients' clinical information and gene expression data(Table 1). Among the various datasets, two major events werespecifically defined: WSB1 levels in metastatic patient samples, and therelationships between WSB1 and HIF target genes. To identifyrelationships between WSB1 and HIF target genes, a classifier describedelsewhere was used (Montagner et al., Nature 487:380-384, 2012).Briefly, a classification rule was defined based on summarizing thestandardized expression levels of WSB1 and HIF-1α's target genes.Analysis of log 2 expression values for both WSB1 and HIF target geneswas carried out using the Prism program.

The relationship between WSB1 expression levels and metastasis freesurvival was defined in human breast cancer and human colon cancerpatient datasets using the PROGgene website (online atwatson.compbio.iupui.edu/chirayu/proggene/database/?url=proggene).

In Vivo Assays for Animal Experiments:

Mice were housed in Specific Pathogen Free (SPF) animal facilities. Formetastasis assays, B16 F10 cells were resuspended in 100 μl of PBS andinjected in the tail vein of C57/B16 male mice, aged-matched between 5and 7 weeks. Seven mice were injected for each sample (3×10⁵ cells foreach mouse). After four weeks, animals were sacrificed and lungs andlivers were removed.

Immunohistochemistry:

Tissue arrays included a lung tumor tissue microarray containing 400pairs of human lung cancer, 21 human invasive colon cancers, and 60human invasive breast cancers, with matched or unmatched normal adjacenttissue. Immunohistochemical staining was performed as describedelsewhere (Yuan et al., supra). Primary antibody dilutions were 1:200for anti-WSB1 (Sigma-Aldrich), and 1:100 for anti-pVHL (SantaCruz).

Example 2—WSB1 Regulates HIF and is Positively Associated withMetastasis in Various Tumors

To determine whether WSB1 has a functional role in tumor aggressiveness,gene expression profiles from 56 pairs of primary lung adenocarcinomapatient samples were analyzed based on WSB1 status, using INGENUITYpathway analysis (FIGS. 2 and 3). Metastasisor migration-related signalssuch as RhoA (Friedl et al., Nature Cell Biol 16:208-210, 2014), TGF-βsignaling (Pickup et al., Nature Rev Cancer 13:788-799, 2013), and actinnucleation by ARP-WASP complex (Bovellan et al., Current Biol24:1628-1635, 2014) are top ranked canonical pathways associated withWSB1 expression (FIG. 2). According to the analysis, WSB1 expression isclosely associated with pathways that are involved in metastasis andinvasion (FIG. 1A). To further investigate the relationship between WSB1and metastasis, a cohort of 83 melanomas, 171 prostate cancers, and 37urinary bladder tumors from three clinically annotated gene-expressiondata sets was analyzed (see, Table 1) for WSB1 expression. In allsamples, WSB1 levels were significantly higher in metastatic tissuesthan in normal or primary tissues (FIG. 1B). Further, for late stagecolon cancer and a subset of breast cancer patients, individuals withhigh WSB1 expression showed lower metastasis-free survival (FIGS. 1C and4A-4C). Interestingly, for breast cancer, WSB1 expression was associatedwith poor survival, mostly in ER-, PR-, HER- or triplenegative-subtypes.

Experiments were then conduced to explore whether or how WSB1 regulatesmetastasis. It was found that endogenous WSB1 expression was positivelycorrelated with the expression of many prometastasis-related genes, andnegatively related with most anti-metastasis genes in lungadenocarcinoma patient samples (Table 2). Moreover, HIF target geneswere closely matched with WSB1 expression level in several cancerpatient samples (FIGS. 1D, 1E, and 5).

WSB1 may be transcriptionally activated by HIF-1α (Benita et al., NuclAcids Res 37:4587-4602, 2009; Tong et al., supra), and this correlationmight reflect a positive correlation among HIF-1α target genes.Interestingly, it was found that HIF-1α levels were greatly increased incells overexpressing WSB1, and decreased in cells depleted of WSB1. WSB1expression in cells depleted of WSB1 restored HIF-1α levels (FIGS. 6A,6B, and 7A), suggesting that WSB1 positively regulates HIF-1α levels.Thus, there may be a feedback loop between WSB1 and HIF-1α. mRNA levelsfor HIF-1α target genes (VEGFA, ALDOC, CA9, and SAP30; Cairns et al.,Cancer 11:85-95, 2011; Gilkes et al., Nature Rev Cancer 14:430-439,2014; and Montagner et al., supra) and HIF-1α were evaluated in cellsoverexpressing WSB1 or depleted of WSB1. WSB1 was found to positivelyregulate mRNA levels of HIF-1α target genes (FIGS. 6C, 6D, and 7B). Incontrast, HIF-1α mRNA levels were not affected by WSB1. These resultssuggested that WSB1 positively regulates HIF-1α levelspost-transcriptionally.

Example 3—WSB1 Regulates pVHL Levels Through Ubiquitinating pVHL

Studies were conducted to examine how WSB1 regulates HIF-1α levels. WSB1was not observed to interact with HIF-1α (FIGS. 8A and 9A). Rather, aninteraction between WSB1 and pVHL was observed (FIGS. 8B and 9A),suggesting that WSB1 might regulate HIF-1α through pVHL. WSB1 is knownas an E3 ligase (Dentice et al., Nature Cell Biol 7:698-705, 2005).Thus, studies were carried out to examine whether pVHL level isregulated by WSB1. When WSB1 was overexpressed, the level of pVHLprotein decreased, and HIF-1α levels increased in a dosage-dependentmanner (FIG. 8C). The decrease in pVHL induced by WSB1 overexpressionwas reversed by the proteasome inhibitor MG 132 (FIG. 8D). Conversely,knocking down WSB1 resulted in a marked increase in pVHL levels (FIG.8E). WSB1 also was found to regulate pVHL stability, as depletion ofWSB1 increased pVHL stability, while reconstitution of cells with WSB1restored pVHL stability to that of control cells (FIG. 8F). Inpancreatic cancer cell lines, NSCLC cell lines, and breast cancer celllines, negative correlations also were observed between WSB1 and pVHLlevels, while positive correlations were observed between WSB1 andHIF-1α (FIG. 9B). These results suggested that WSB1 negatively regulatespVHL stability, possibly through regulation of pVHL ubiquitination.Indeed, knocking down WSB1 was found to decrease pVHL ubiquitination(FIG. 8G).

Example 4—the SOCS Domain of WSB1 is Required for pVHL Interaction andUbiquitination

WSB1 contains WD repeats and a SOCS domain (Dentice et al., supra). Todetermine the regions of WSB1 that are required for its interaction withpVHL, Myc-tagged WSB1 deletion mutants were expressed in cells, andco-immunoprecipitation was performed. These studies showed that theinteraction of WSB1 with pVHL requires the SOCS domain. In addition, thedown regulation of pVHL by WSB1 was found to be dependent on the SOCSdomain (FIGS. 10A and 10B). WSB1 fragments containing the SOCS domainled to decreased pVHL levels, while fragments lacking the SOCS domaincould not do so (FIG. 10B). Further, reconstituting cells depleted ofWSB1 with WT WSB1 restored pVHL polyubiquitination, while reconstitutingthese cells with WSB1 (Δ SOCS) did not (FIG. 10C). WSB1 also interacteddirectly with pVHL in vitro (FIG. 10D), and promoted pVHLpolyubiquitination in vitro (FIG. 10E). Thus, WBS1 appears to be is anE3 ligase that regulates pVHL ubiquitination in cells.

Example 5—WSB1 Promotes pVHL Proteasomal Degradation in Hypoxia

Given that WSB1 regulates pVHL and HIF-1α levels under normoxicconditions, studies were conducted to assess whether WSB1 also regulatespVHL and HIF-1α levels under hypoxia condition. HIFs are unstableproteins that are degraded even under hypoxic conditions (Kong et al., JBiol Chem 282:15498-15505, 2007; Liu et al., Oncogene 30:21-31, 2011;and Uchida et al., J Biol Chem 279:14871-14878, 2004). The interactionbetween WSB1 and pVHL was increased under hypoxia and hypoxia mimicconditions (FIGS. 11A and 12A). This increase might be due to increasedWSB1 expression under hypoxic conditions. In addition, when cells werecultured under hypoxic conditions, endogenous pVHL levels decreasedwhile HIF-1α, HIF-2α, and WSB1 levels increased (FIGS. 11B and 12B).Interestingly, VHL mRNA levels did not changed in hypoxia, while forcomparison HIF-1α, Glut1 (an HIF-1α target gene) and WSB1 mRNA levelsincreased under the same conditions (FIG. 11B), supporting the idea thatWSB1 regulates pVHL at the post-transcriptional level, and acts as an E3ubiquitin ligase for pVHL in hypoxia.

Further, ubiquitination of pVHL was increased under hypoxia-mimicconditions, and knocking down WSB1 decreased pVHL polyubiquitination(FIG. 11C). Knocking down WSB1 also decreased the up regulation ofHIF-1α under hypoxia conditions (FIG. 11D). Overall, these resultssuggested that WSB1 regulates pVHL turnover and contributes to HIF-1αand HIF-2α up regulation under hypoxic conditions.

Example 6—WSB1 Promotes Cancer Cell Invasion, Migration and Metastasisby Inhibiting pVHL

To determine whether WSB1 upregulates HIF-1α levels and promotes tumormetastasis by down regulating pVHL, RCC4 and 786-O renal carcinoma celllines lacking pVHL were used, as were their derivatives reconstitutedwith HA-pVHL (RCC4/VHL and 786-O/VHL; Li et al., Mol Cell Biol27:5381-5392, 2007). These studies showed that overexpression of WSB1had no effect on HIF-1α and HIF-2α levels in VHL-deficient RCC4 and786-O cells (FIGS. 13A and 14A). WSB1 was able to increase HIF-1α levelsonly in cells reconstituted with pVHL. Overexpression of WSB1 had nosignificant effect on cell invasiveness and mobility in VHL-deficientRCC4 and 786-O cells (FIGS. 13B, 13C, and 14B-14D), while substantiallyincreasing cell motility and invasiveness of cells reconstituted withpVHL. Further, depletion of WSB1 decreased HIF-1α activity only inRCC4/VHL cells and 786-O/VHL cells, but did not affect HIF-1α activityin RCC4 and 786-O cells (FIG. 13D). Reconstitution of cells with WT butnot WSB1 ΔSOCS in cells depleted of WSB1 restored HIF-1α levels andactivity (FIG. 13D). Similar results were obtained using the breastcancer cell line MDA-MB 231 (FIGS. 14E-14G). These results suggestedthat WSB1 regulates HIF, cell motility, and cell invasion through pVHL.

WSB1 can down regulate HIPK2 through its E3 ligase activity (Choi etal., supra; and Tong et al., supra). Because HIPK2 regulates multiplecellular processes, including p53 activation (Jin et al., Nature Med18:580-588, 2012; Puca et al., supra; and Rinaldo et al., Molecular Cell47:87-98, 2012), experiments were carried out to test whether HIPK2regulation by WSB1 plays a role in HIF-1α activity and cell mobility. Asshown in FIG. 15, WSB1 overexpression was able to increase HIF-1α levelsand cell mobility in cells depleted of HIPK2, suggesting that WSB1regulates HIF-1α in a HIPK2 independent manner.

To investigate the functional relevance of the WSB1-pVHL axis inmalignant behavior in vivo, cellular metastasis assays were conducted invivo in mouse models. Depletion of WSB1 resulted in significantlydecreased lung and liver colonization of B16F10 melanoma cells aftertail-vein injection. These effects were reversed by reconstitution of WTWSB1 but not WSB1 ΔSOCS (FIGS. 16A-16C). Thus, WSB1 appears to beimportant for metastasis in malignant tumors through pVHL degradationand HIF-1α up regulation.

Finally, to examine the expression of WSB1 and pVHL in human tumortissue, immuno-histochemical staining of WSB1 and pVHL was performed in400 lung cancer specimens spotted on a tissue microarray (FIGS. 17A and17B), and on 60 metastatic breast and 21 metastatic colorectal carcinomapatients' slides (FIG. 16D). WSB1 expression was higher in cancerlesions compared to normal adjacent tissues, while pVHL expressiongenerally was negatively correlated with WBS1. These results areconsistent with the negative regulation of pVHL by WSB1 in humancancers.

TABLE 1 #samples Cancer Type Data set Platform Series Total MetastasisMelanoma GDS3966 GPL96 GSE840 83 50 Prostate GDS2545 GPL8300 GSE6916 17125 Synchronous and GDS3501 GPL570 GSE10961 18 18 metachronous livermetastases from colorectal cancer Carcinoma in situ GDS1479 GPL96GSE3167 37 13 lesions of the urinary bladder Breast GDS3096 GPL96GSE5847 47 34

TABLE 2 Fold-Change Genes in p-value (WSB1 High Expression vs ID dataset(Wsb1 status) WSB1 Low expression) CXCR4 CXCR4   9.29E−08 2.365 AKT2AKT2 3.50594E−07 2.241 PTCH PTCH1   1.36E−07 2.184 MMP11 MMP11 0.0197392.168 HGF HGF 2.24632E−10 2.128 FABP5 FABP5  3.0097E−07 2.069 IL8RBCXCR2   4.12E−06 2.042 EGF EGF 2.83822E−06 2.026 CD274 CD274 5.04073E−081.985 SLC7A11 SLC7A11 0.000904253 1.980 CUTL1 CUX1   1.07E−09 1.933ANGPT2 ANGPT2 0.0012662 1.918 PGF PGF 0.00637047 1.891 PPM1D PPM1D9.39074E−09 1.875 ZAP70 ZAP70 1.48671E−08 1.844 MMP9 MMP9   3.54E−051.840 CA4 CA4 0.011951 1.782 ITGB3 ITGB3 9.60728E−05 1.768 VEGFA VEGFA  1.14E−05 1.749 NR4A1 NR4A1 0.008555 1.716 TACSTD1 EPCAM 2.53482E−061.702 NUAK1 NUAK1 1.26095E−07 1.693 LGALS3 LGALS3 3.68585E−10 1.686 HK2HK2   3.52E−06 1.683 TGFB2 TGFB2 0.000162424 1.677 SLC38A6 SLC38A62.54046E−09 1.667 NT5E NT5E 4.88986E−05 1.651 CCR4 CCR4 0.0029205 1.645HTATIP2 HTATIP2 3.39708E−05 1.839 OLFML3 OLFML3 6.39708E−07 1.617 ANK3ANK3 1.29437E−07 1.609 CSF1 CSF1   2.05E−06 1.602 SP4 SP4 3.76181E−091.599 CAPN2 CAPN2 6.11261E−08 1.595 SLC16A3 SLC16A3 8.32484E−06 1.591DLG7 DLGAP5 0.00644031 1.586 CYP1B1 CYP1B1 6.01141E−07 1.580 ESR1 ESR10.000711501 1.574 TUBE1 TUBE1 2.74138E−07 1.572 NDRG1 NDRG1   2.12E−051.565 MET MET 0.000294532 1.561 MMP2 MMP2   3.50E−08 1.556 TGFA TGFA0.000114944 1.552 TUBD1 TUBD1 5.81612E−08 1.550 CAT CAT 2.38149E−081.545 AKR1C3 AKR1C3 0.000215814 1.530 APC APC 1.51751E−07 1.520 CFLARCFLAR  8.531E−11 1.519 TNIK TNIK 0.00117395 1.515 SELE SELE 0.0003739481.512 TUBB2C TUBB4B 2.88577E−07 1.509 ANGPTL4 ANGPTL4 2.32619E−05 1.505GREM2 GREM2 0.0113608 −1.519 ING4 ING4   7.52E−09 −1.519 PPARD PPARD  5.90E−05 −1.530 STAB2 STAB2 0.0215461 −1.530 IL1RL1 IL1RL1 0.0191785−1.547 CLDN3 CLDN3 0.000603823 −1.559 LGR4 LGR4 0.00774795 −1.581 REV3LREV3L   3.55E−05 −1.634 DFFB DFFB 4.21524E−07 −1.637 NLRP1 NLRP1  3.05E−05 −1.669 NFKB1 NFKB1   7.13E−13 −1.700 WWOX WWOX 6.82828E−11−1.714 KISS1 KISS1 0.00311776 −1.732 ISLR ISLR 0.000414443 −1.735 SDC1SDC1   3.37E−07 −1.792 CDCA7L CDCA7L   7.02E−05 −1.801 CXCR7 CXCR70.000331741 −1.808 TP53 TP53   1.86E−05 −1.817 ANPEP ANPEP 0.0115719−1.847 LTB4R2 LTB4R2   6.01E−08 −1.868 TP73L TP63 0.0133901 −1.868 CNN1CNN1 0.000290524 −1.917 PSCA PSCA 0.0203717 −1.934 ZNF350 ZNF3502.81979E−08 −1.946 CDKN2C CDKN2C   4.27E−11 −1.986 DSG2 DSG2   8.64E−06−1.997 PPARA PPARA   6.32E−12 −2.024 LTF LTF 0.000635644 −2.156 CCNA1CCNA1   1.16E−05 −2.157 C1R C1R   3.95E−08 −2.272 MSC MSC   5.97E−07−2.358 NF2 NF2   8.12E−11 −2.454

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a cancer patient,comprising administering to the cancer patient an agent that reduces theactivity of WSB1, wherein the cancer is melanoma.
 2. The method of claim1, wherein the agent is an inhibitory nucleic acid targeted to a WSB1nucleic acid that is endogenous to the cancer patient.
 3. The method ofclaim 2, wherein the inhibitory nucleic acid is a shRNA.
 4. The methodof claim 1, comprising administering to the cancer patient a compositioncomprising the agent and a pharmaceutically acceptable carrier.
 5. Amethod for inhibiting metastasis of a tumor in a cancer patient,comprising administering to the cancer patient an agent that reduces theactivity of WSB1, wherein the cancer is melanoma.
 6. The method of claim5, wherein the agent is an inhibitory nucleic acid targeted to a WSB1nucleic acid that is endogenous to the cancer patient.
 7. The method ofclaim 6, wherein the inhibitory nucleic acid is a shRNA.
 8. The methodof claim 5, comprising administering to the cancer patient a compositioncomprising the agent and a pharmaceutically acceptable carrier.